Oxygenolysis reaction mechanism of copper-dependent quercetin 2,3-dioxygenase: A density functional theory study

The mechanism of the action of copper-dependent quercetin 2,3-dioxygenase(2,3QD) has been investigated by means of hybrid density functional theory.The 2,3QD enzyme cleaves the O-heterocycle of a quercetin by incorporation of both oxygen atoms into the substrate and releases carbon monoxide.The calculations show that dioxygen attack on the copper complex is energetically favorable.The adduct has a possible near-degeneracy of states between [Cu 2+-(substrate-H +)] and [Cu +-(substrate-H).],and in addition the pyramidalized C 2 atom is ideally suited for forming a dioxygen-bridged structure.In the next step,the C 3-C 4 bond is cleaved and intermediate Int 5 is formed via transition state TS 4.Finally,the O a-O b and C 2-C 3 bonds are cleaved,and CO is released in one concerted transition state(TS 5) with the barrier of 63.25 and 61.91 kJ/mol in the gas phase and protein environments,respectively.On the basis of our proposed reaction mechanism,this is the rate-limiting step of the whole catalytic cycle and is strongly driven by a relatively large exothermicity of 100.86 kJ/mol.Our work provides some valuable fundamental insights into the behavior of this enzyme.     

Density functional theory study on a missing piece in understanding of heme chemistry: the reaction mechanism for indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme-containing dioxygenases and catalyze oxidative cleavage of the pyrrole ring of L-tryptophan. On the basis of three recent crystal structures of these heme-containing dioxygenases, two new mechanistic pathways were proposed by several groups. Both pathways start with electrophilic addition of the Fe(II)-bound dioxygen concerted with proton transfer (oxygen ene-type reaction), followed by either formation of a dioxetane intermediate or Criegee-type rearrangement. However, density functional theory (DFT) calculations do not support the proposed concerted oxygen ene-type and Criegee-type rearrangement pathways. On the basis of DFT calculations, we propose a new mechanism for dioxygen activation in these heme systems. The mechanism involves (a) direct electrophilic addition of the Fe(II)-bound oxygen to the C2 or C3 position of the indole in a closed-shell singlet state or (b) direct radical addition of the Fe(III)-superoxide to the C2 position of the indole in a triplet (or open-shell singlet) state. Then, a radical-recombination or nearly barrierless charge-recombination step from the resultant diradical or zwitterionic intermediates, respectively, proceeds to afford metastable dioxetane intermediates, followed by ring-opening of the dioxetanes. Alternatively, homolytic O-O bond cleavage from the diradical intermediate followed by oxo attack and facile C2-C3 bond cleavage could compete with the dioxetane formation pathway. Effects of ionization of the imidazole and negatively charged oxyporphyrin complex on the key dioxygen activation process are also studied.     

Hybrid DFT study of the mechanism of quercetin 2,3-dioxygenase

The mechanism of the copper-containing enzyme quercetin 2,3-dioxygenase has been studied using hybrid density functional theory. This enzyme cleaves the O-heterocycle of a flavonol using dioxygen and releases carbon monoxide. Two different pathways for the dioxygen attack on the copper complex have been investigated, and the one where the first attack is on copper is found to be the energetically preferred one. By using this pathway the problem of having to go through a spin-orbit-induced spin crossing is also avoided. The adduct has three unpaired spins and is ideally suited for forming a dioxygen bridging structure, which occurs in the next step. Rather than cleaving the O-O bond in the next step, another C-O bond between dioxygen and the substrate is first formed. Finally, the O-O bond is cleaved, and CO is released in one concerted transition state with a very low barrier. The results are in good agreement with experimental findings. The mechanism is compared to the ones for other similar enzymes studied recently by similar methods.     

Theoretical study of the energetics of the reactions of triplet dioxygen with hydroquinone, semiquinone, and their protonated forms: relation to the mechanism of superoxide generation in the respiratory chain

One-electron reduction of the dioxygen molecule by the reduced form of mitochondrial ubiquinones (Q) of the NADH dehydrogenase (complex I) and mitochondrial cytochrome bc1 (complex III) is believed to be the main source of the superoxide anion radical O2*- and the hydroperoxide radical OOH*. In this work, we modeled the energetics of four possible reactions of the triplet ((3)Sigma(g)) dioxygen-molecule reduction by fully reduced and protonated ubiquinone (QH2; reaction 1), its deprotonated form (QH-; reaction 2), the semiquinone radical (QH*; reaction 3), and the semiquinone anion radical (Q*-; reaction 4), by means of ab initio calculations with the 6-31G(d) and 6-31+G(d) basis set in the restricted open-shell Hartree-Fock (ROHF), unrestricted Hartree-Fock (UHF), and complete active space self-consistent field (CASSCF) with dynamic correlation [at the second-order M?ller-Plesset (MP2) or multiple reference M?ller-Plesset (MRMP), respectively] schemes and the basis set superposition error (BSSE) correction included, as well as semiempirical AM1 and PM3 calculations in the UHF and ROHF schemes. 2-Butene-1,4-dione and p-benzoquinone were selected as model compounds. For the reduced forms of both compounds, reaction 1 turned out to be energetically unfavorable at all levels of theory, this agreeing with the experimentally observed diminished reductive properties of hydroquinone derivatives at low pH. For 2-butene-1,4-dione treated at the most advanced MRMP/CASSCF/6-31+G(d) level, the energies of reactions 1-4 are 4.7, -34.3, -15.0, and -4.1 kcal/mol, respectively. This finding suggests that reactions 2 and 3 are the most likely mechanisms of electron transfer to molecular oxygen in aprotic environments and that proton transfer is involved in this process. Nearly the same energies of reactions 2 and 3 were calculated at the MRMP/CASSCF/6-31+G(d) level for reduced forms of p-benzoquinone. Inclusion of diffuse functions in the basis set and dynamic correlation at the CASSCF level appears essential. Because deprotonated ubiquinol is unlikely to exist in physiological environments, reaction 3 appears to be the most likely mechanism of one-electron reduction of oxygen; however, if oxygen can penetrate cytochrome bc1 as far as the Q(o) center where ubiquinol can be deprotonated, reaction 2 can also come into play. The energies of reactions 2 and 3 calculated at the MRMP/CASSCF/6-31+G(d) level are most closely reproduced in the ab initio and semiempirical UHF PM3 calculations. Additional semiempirical calculations on more realistic models of ubiquinone, 2,3-dimethoxy-6-methyl-p-benzoquinone and 2,3-dimethoxy-5-isoprenyl-6-methyl-p-benzoquinone, gave qualitatively the same relations between the energies of reactions 2 and 3 as those carried out for p-benzoquinone species, thereby suggesting that this method could be used in studying electron-transfer reactions from reduced quinone derivatives to molecular oxygen in more complex systems, such as a model of the Q(o) site of cytochrome bc1, where applying ab initio methods is unfeasible.     

Roles of radical characters of pristine and nitrogen-substituted hydrographene in dioxygen bindings

We investigate by means of density functional theory (DFT) calculations how hydrogen-terminated graphenes (hydrographenes) with and without nitrogen impurities interact with dioxygen. The current study aims at searching whether hydrographenes can be utilized as cathode catalysts in fuel cell with a focus on dioxygen binding, the first step in oxygen reduction reaction (ORR). If hydrographenes have a nanometer-size rhombic structure with zigzag edges, unpaired electrons are localized at their edges with or without the nitrogen impurities. Spin localization comes from frontier orbitals of the nanometer-size hydrographenes whose amplitudes appear only at their edges. Due to their radical characters, dioxygen can bind to an edge carbon atom of the hydrographenes under the condition where fuel cell is usually operated. There are two types of dioxygen binding into a hydrographene: one is a Pauling fashion where one C-O bond is formed and the other is a bridging fashion with two formed C-O bonds. In the bridging fashion, the formation of the two C-O bonds activates dioxygen, and then radical characters of the oxygen atoms completely disappear. In contrast, the Pauling fashions retain an unpaired electron on the oxygen atom that does not participate to the C-O bond formation. The existence of radical oxygen atoms would facilitate the next step in ORR (the initial proton transfer to an adsorbed dioxygen), whereas such facilitative effects cannot be seen in its absence. According to DFT calculations, the Pauling-type bindings are always energetically preferred over the bridging-type bindings. In particular, the C→N substitution enhances the preferences of the Pauling-type binding over the bridging-type binding compared with the pristine case. Accordingly DFT calculations demonstrate that radical characters of edge carbons of a nanometer-sized rhombic hydrographene play a crucial role in dioxygen bindings in a Pauling fashion that would be responsible for enhancing the catalytic activity in fuel cell.     

Human tryptophan dioxygenase: a comparison to indoleamine 2,3-dioxygenase

In contrast to the diverse superfamily of monooxygenases, there are only two classes of heme-containing dioxygenases in humans. One is tryptophan 2,3 dioxygenase (hTDO), and the other is indoleamine 2,3-dioxygenase (hIDO), both of which catalyze the oxidative degradation of Trp to N-formyl kynurenine. Although hTDO and hIDO catalyze the same reaction, they engage in distinct physiological functions. The molecular properties of hTDO, unlike hIDO, have never been explored in the past. Here, we report the first structural and functional characterization of hTDO with resonance Raman and optical absorption spectroscopies. We show that the proximal Fe-His stretching frequency of hTDO is 229 cm(-1), 7 cm(-1) lower than that of hIDO, indicating its weaker imidazolate character as compared to hIDO. In the CO derivative of the L-Trp-bound enzyme, the Fe-CO stretching and C-O stretching frequencies are 488 and 1972 cm(-1), respectively, suggesting that L-Trp binds to the distal pocket with its C2-C3 double bond facing the heme-bound ligand, in contrast to hIDO, in which the indole NH group forms an H-bond with the heme-bound ligand. Moreover, the Km values of hTDO for D-Trp and L-Trp are similar, but the kcat value for D-Trp is 10-fold lower than that for L-Trp. In contrast, in hIDO, the Km value for D-Trp is 700-fold higher than L-Trp, whereas the kcat values are comparable for the two stereoisomers. Taken together, the data indicate that the initial deprotonation reaction of the indole NH group in hTDO is carried out by the evolutionarily conserved distal His, whereas that in hIDO is performed by the heme-bound dioxygen; in addition, the stereospecificity of hTDO is determined by the efficiency of the dioxygen chemistry, whereas that in hIDO is controlled by the substrate affinity.     

Study of the reaction products of flavonols with 2,2-diphenyl-1-picrylhydrazyl using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry

The products obtained after the reaction between flavonols and the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) in both methanol and acetonitrile were characterized using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and NMR spectroscopy. The flavonols studied were quercetin, kaempferol and myricetin. In methanol, two reaction products of oxidized quercetin were identified using LC/ESI-MS/MS and NMR. Quercetin was oxidized through a transfer of two H-atoms to DPPH(*) and subsequently incorporated either two CH(3)OH molecules or one CH(3)OH- and one H(2)O molecule giving the products 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dimethoxy-2,3-dihydrochromen-4-one and 2-(3,4-dihydroxyphenyl)-3,3,5,7-tetrahydroxy-2-methoxy-2,3-dihydrochromen-4-one, respectively. LC/ESI-MS/MS analysis revealed that in methanol, kaempferol and myricetin also gave rise to methoxylated oxidation products similar to that identified for quercetin. Kaempferol, in addition, also exhibited products where a kaempferol radical, obtained by a transfer of one H-atom to DPPH(*), reacted with CH(3)OH through the addition of CH(3)O(*), yielding two isomeric products. When the reaction took place in acetonitrile, LC/ESI-MS/MS analysis showed that both quercetin and myricetin formed stable isomeric quinone products obtained by a transfer of two H-atoms to DPPH(*). In contrast, kaempferol formed two isomeric products where a kaempferol radical reacted with H(2)O through the addition of OH(*), i.e. similar to the reaction of kaempferol radicals with CH(3)OH.     

Common system setup for the entire catalytic cycle of cytochrome P450(cam) in quantum mechanical/molecular mechanical studies

We describe a system setup that is applicable to all species in the catalytic cycle of cytochrome P450(cam). The chosen procedure starts from the X-ray coordinates of the ferrous dioxygen complex and follows a protocol that includes the careful assignment of protonation states, comparison between different conceivable hydration schemes, and system preparation through a series of classical minimizations and molecular dynamics (MD) simulations. The resulting setup was validated by quantum mechanical/molecular mechanical (QM/MM) calculations on the resting state, the pentacoordinated ferric and ferrous complexes, Compound I, the transition state and hydroxo intermediate of the C--H hydroxylation reaction, and the product complex. The present QM/MM results are generally consistent with those obtained previously with individual setups. Concerning hydration, we find that saturating the protein interior with water is detrimental and leads to higher structural flexibility and catalytically inefficient active-site geometries. The MD simulations favor a low water density around Asp251 that facilitates side chain rotation of protonated Asp251 during the conversion of Compound 0 to Compound I. The QM/MM results for the two preferred hydration schemes (labeled SE-1 and SE-4) are similar, indicating that slight differences in the solvation close to the active site are not critical as long as camphor and the crystallographic water molecules preserve their positions in the experimental X-ray structures.     

The final catalytic step of cytochrome p450 aromatase: a density functional theory study

B3LYP density functional theory calculations are used to unravel the mysterious third step of aromatase catalysis. The feasibility of mechanisms in which the reduced ferrous dioxygen intermediate mediates androgen aromatization is explored and determined to be unlikely. However, proton-assisted homolysis of the peroxo hemiacetal intermediate to produce P450 compound I and the C19 gem-diol likely proceeds with a low energetic barrier. Mechanisms for the aromatization and deformylation sequence which are initiated by 1beta-hydrogen atom abstraction by P450 compound I are considered. 1beta-Hydrogen atom abstraction from substrates in the presence of the 2,3-enol encounters strikingly low barriers (5.3-7.8 kcal/mol), whereas barriers for this same process rise to 17.0-27.1 kcal/mol in the keto tautomer. Transition states for 1beta-hydrogen atom abstraction from enolized substrates in the presence of the 19-gem-diol decayed directly to the experimentally observed products. If the C19 aldehyde remains unhydrated, aromatization occurs with concomitant decarbonylation and therefore does not support dehydration of the C19 aldehyde prior to the final catalytic step. On the doublet surface, the transition state connects to a potentially labile 1(10) dehydrogenated product, which may undergo rapid aromatization, as well as formic acid. Ab initio molecular dynamics confirmed that the 1beta-hydrogen atom abstraction and deformylation or decarbonylation occur in a nonsynchronous, coordinated manner. These calculations support a dehydrogenase behavior of aromatase in the final catalytic step, which can be summarized by 1beta-hydrogen atom abstraction followed by gem-diol deprotonation.     

Computational and spectroscopic characterization of the molecular and electronic structure of the Pb(II)-quercetin complex

The interactions of lead(II) ion with a polyhydroxylated flavonoid, the quercetin molecule, were investigated in methanol solution. The quercetin/metal stoichiometries and equilibrium stability constants for metal binding to quercetin have been determined by UV-vis spectroscopy combined with chemometrics methods. The 2:1, 1:2, and predominant 1:1 species are formed in solution. Among the three potential sites of chelation present in the quercetin structure, the catechol function presents the highest complexation power toward Pb(II), in opposition with previous results found for Al(III) complexation. This result has been confirmed by the good agreement of the experimental and theoretical features for both the electronic and vibrational spectra of the 1:1 complex. DT-DFT calculations show that the bathochromic shift of the long-wavelength band of the UV-vis spectra, that occurs upon complexation, is due to a ligand-to-metal charge transfer. The molecular structure of the ligand is not much modified by the coordination of lead at the level of the catecholate.     

Photolytic reactions of subvalent aluminum(I) halides in the presence of dioxygen: generation and characterization of the peroxo species XAlO(2) and XAl(mu-O)(2)AlX (X = F,Cl, Br)

The photolytic reactions of AlX (X = F, Cl, and Br) with O(2) in solid argon matrixes are shown to yield the peroxo species XAlO(2), all exhibiting C(2)(v)() symmetry. The species were identified and characterized by means of IR spectroscopy allied with quantum mechanical calculations. In addition to singlet XAlO(2) as the main product of the reaction of AlX, the experiments give clear evidence for the formation of XAl(mu-O)(2)AlX, by the reaction of the dimer (AlX)(2), which is also known to be present in the matrixes upon deposition. Finally, weak IR absorptions were tentatively assigned to XAlO(2) in its triplet electronic state. According to our calculations, the singlet-triplet gap amounts to about 40 kJ mol(-1) for all species. The properties of the peroxide species invite comparison with previously investigated dioxygen complexes, as well as the superoxide species XAlOO and various possible products of the reaction of (AlX)(2) dimers.     

Selective response of mesoporous silicon to adsorbants with nitro groups

We demonstrate that the electronic structure of mesoporous silicon is affected by adsorption of nitro-based explosive molecules in a compound-selective manner. This selective response is demonstrated by probing the adsorption of two nitro-based molecular explosives (trinitrotoluene and cyclotrimethylenetrinitramine) and a nonexplosive nitro-based aromatic molecule (nitrotoluene) on mesoporous silicon using soft X-ray spectroscopy. The Si atoms strongly interact with adsorbed molecules to form Si-O and Si-N bonds, as evident from the large shifts in emission energy present in the Si L(2,3) X-ray emission spectroscopy (XES) measurements. Furthermore, we find that the energy gap (band gap) of mesoporous silicon changes depending on the adsorbant, as estimated from the Si L(2,3) XES and 2p X-ray absorption spectroscopy (XAS) measurements. Our ab initio molecular dynamics calculations of model compounds suggest that these changes are due to spontaneous breaking of the nitro groups upon contacting surface Si atoms. This compound-selective change in electronic structure may provide a powerful tool for the detection and identification of trace quantities of airborne explosive molecules.     

Energetics of dioxygen binding into graphene patches with various sizes and shapes

Density functional theory (DFT) calculations were employed to investigate the electronic properties of an H-atom terminated graphene patch (hydrographene) smaller than a rhombic C96H26 structure with zigzag edges. Depending on shapes and sizes of hydrographenes, some hydrographenes have the triplet ground state where unpaired electrons are localized on their zigzag edges. The stability of the triplet spin state is diminished, decreasing the hydrographene sizes. The existence of the localized spin densities allows triplet dioxgen to bind into a hydrographene. According to the DFT calculations, the energetics of the dioxygen bindings is negatively influenced by downsizing hydrographenes, as well as depends on their shapes. The size-and shape-dependences of the dioxygen bindings reflect from the stability of the triplet state of a hydrographene, because its localized unpaired electrons can be utilized to be attached to an unpaired electron of triplet dioxygen.     

Mechanistic Insights into a Stibene Cleavage Oxygenase NOV1 from Quantum Mechanical/Molecular Mechanical Calculations

NOV1, a stilbene cleavage oxygenase, catalyzes the cleavage of the central double bond of stilbenes to two phenolic aldehydes, using a 4-His Fe(II) center and dioxygen. Herein, we use in-protein quantum mechanical/molecular mechanical (QM/MM) calculations to elucidate the reaction mechanism of the central double bond cleavage of phytoalexin resveratrol by NOV1. Our results showed that the oxygen molecule prefers to bind to the iron center in a side-on fashion, as suggested from the experiment. The quintet Fe−O₂ complex with the side-on superoxo antiferromagnetic coupled to the resveratrol radical is identified as the reactive oxygen species. The QM/MM results support the dioxygenase mechanism involving a dioxetane intermediate with a rate-limiting barrier of 10.0 kcal mol⁻¹. The alternative pathway through an epoxide intermediate is ruled out due to a larger rate-limiting barrier (26.8 kcal mol⁻¹). These findings provide important insight into the catalytic mechanism of carotenoid cleavage oxygenases and also the dioxygen activation of non-heme enzymes.     

On the Metal Cooperativity in a Dinuclear Copper–Guanidine Complex for Aliphatic C−H Bond Cleavage by Dioxygen

Selective oxidation reactions of organic compounds with dioxygen using molecular copper complexes are of relevance to synthetic chemistry as well as enzymatic reactivity. In the enzyme peptidylglycine α-hydroxylating monooxygenase (PHM), the hydroxylating activity towards aliphatic substrates arises from the cooperative effect between two copper atoms, but the detailed mechanism has yet to be fully clarified. Herein, we report on a model complex showing hydroxylation of an aliphatic ligand initiated by dioxygen. According to DFT calculations, the proton-coupled electron-transfer (PCET) process leading to ligand hydroxylation in this complex benefits from cooperative effects between the two copper atoms. While one copper atom is responsible for dioxygen binding and activation, the other stabilizes the product of intramolecular PCET by copper–ligand charge transfer. The results of this work might pave the way for the directed utilization of cooperative effects in oxidation reactions.     

Formation and Characterization of ZrO3 and HfO3 Molecules in Solid Argon

ZrO3 and HfO3 molecules were prepared via reactions of metal monoxides with dioxygen in solid argon and were characterized using matrix isolation infrared absorption spectroscopy as well as theoretical calculations. Unlike the titanium monoxide molecule, which reacted spontaneously with dioxygen to form TiO3, the ZrO and HfO molecules reacted with dioxygen to give the ZrO3 and HfO3 molecules only under visible light irradiation. Density functional calculations predicted that both the ZrO3 and HfO3 molecules possess a closed-shell singlet ground state with a non-planar C8 geometry, in which the side-on coordinated O2 falls into the peroxide category.     

Reactivity of Cl–P+–Cl toward cyclic organic ethers

The dichlorophosphenium ion (Cl-P(+)-Cl) undergoes a variety of reactions with cyclic organic ethers in the gas phase in a Fourier-transform ion cyclotron resonance mass spectrometer. Most of the reactions are initiated by Cl-P(+)-Cl-induced heterolytic C-O bond cleavage. However, the observed final products depend on the exact structure of the ether. For saturated ethers, e.g., tetrahydropyran, tetrahydrofuran, and 2-methyltetrahydrofuran, the most abundant ionic product corresponds to hydroxide abstraction by Cl-P(+)-Cl. This unexpected reaction is rationalized by a multistep mechanism that involves an initial heterolytic C-O bond cleavage accompanied by a 1,2-hydride shift, and that ultimately yields a resonance-stabilized allyl cation and HOPCl2. The process is estimated to be highly exothermic (AM1 calculations yield delta H = -(33-38) kcal mol(-1) for the ethers mentioned above). However, the adducts formed from most of the unsaturated ethers are unable to undergo hydride shifts and hence cannot react via this pathway. In some of these cases, e.g., for 2,5-dihydrofuran and 2,5-dihydro-3,4-benzofuran, the C-O bond heterolysis is followed by oxygen/chlorine exchange to yield the O=PCl radical and a resonance-stabilized carbocation (AM1 calculations yield delta H = -14 kcal mol(-1) for the reaction of 2,5-dihydro-3,4-benzofuran). Hydride abstraction by Cl-P(+)-Cl also yields an abundant product for these two ethers. On the other hand, the ethers with low ionization energies, such as 2,3-dihydrofuran and 2,3-dihydrobenzofuran, react with Cl-P(+)-Cl by electron transfer. Finally, a unique pathway, addition followed by elimination of HCl, dominates the reaction with furan. The observed reactions are rationalized by thermochemical data obtained from semiempirical molecular orbital calculations.     

Theoretical clues to the mechanism of dioxygen formation at the oxygen-evolving complex of photosystem II

The mechanism of the generation of dioxygen at the oxygen-evolving complex (OEC) of photosystem II (PSII), a crucial step in photosynthesis, is still under debate. The simplest unit present in the OEC that can produce O2 is a dinuclear oxo-bridge manganese complex within the tetranuclear Mn4 cluster. In this paper we report a theoretical study of the model complexes [Mn2(mu-O)2(NH3)6(H2O)2]n+ (n = 2-5), for which density functional calculations have been carried out for several electronic configurations. The molecular orbital picture deduced from the calculations indicates that one-electron oxidation of the Mn2IV,IV/(O2-)2 complex (n = 4) mostly affects the oxygen atoms, thus ruling out the existence of a MnV oxidation state in this context, while the incipient formation of an O-O bond in the O2(3-) transient species evolves exothermally toward the dissociation of dioxygen and a Mn2II,III couple. These results identify the electronic features that could be needed to enable an intramolecular mechanism of oxygen-oxygen bond formation to exist at the OEC during photosynthesis.